ChemEM: Flexible Docking of Small Molecules in Cryo-EM Structures.

Cryo-electron microscopy (cryo-EM), through resolution advancements, has become pivotal in structure-based drug discovery. However, most cryo-EM structures are solved at 3-4 Å resolution, posing challenges for small-molecule docking and structure-based virtual screening due to issues in the precise positioning of ligands and the surrounding side chains. We present ChemEM, a software package that employs cryo-EM data for the accurate docking of one or multiple ligands in a protein-binding site. Validated against a highly curated benchmark of high- and medium-resolution cryo-EM structures and the corresponding high-resolution controls, ChemEM displayed impressive performance, accurately placing ligands in all but one case, often surpassing cryo-EM PDB-deposited solutions. Even without including the cryo-EM density, the ChemEM scoring function outperformed the well-established AutoDock Vina score. Using ChemEM, we illustrate that valuable information can be extracted from maps at medium resolution and underline the utility of cryo-EM structures for drug discovery.

New prediction categories in CASP15.

Prediction categories in the Critical Assessment of Structure Prediction (CASP) experiments change with the need to address specific problems in structure modeling. In CASP15, four new prediction categories were introduced: RNA structure, ligand-protein complexes, accuracy of oligomeric structures and their interfaces, and ensembles of alternative conformations. This paper lists technical specifications for these categories and describes their integration in the CASP data management system.

Stoichiometry-Selective Antagonism of α4ß2 Nicotinic Acetylcholine Receptors by Fluoroquinolone Antibiotics.

Quinolone antibiotics disrupt bacterial DNA synthesis by interacting with DNA gyrase and topoisomerase IV. However, in addition, they have been shown to act as inhibitors of pentameric ligand-gated ion channels such as GABAA receptors and the α7 nicotinic acetylcholine receptor (nAChR). In the present study, we have examined the effects of quinolone antibiotics on the human α4ß2 nAChR, an important subtype that is widely expressed in the central nervous system. A key feature of α4ß2 nAChRs is their ability to coassemble into two distinct stoichiometries, (α4)2(ß2)3 and (α4)3(ß2)2, which results in differing affinities for acetylcholine. The effects of nine quinolone antibiotics were examined on both stoichiometries of the α4ß2 receptor by two-electrode voltage-clamp recording. All compounds exhibited significant inhibition of α4ß2 nAChRs. However, all of the fluoroquinolone antibiotics examined (ciprofloxacin, enoxacin, enrofloxacin, difloxacin, norfloxacin, pefloxacin, and sparfloxacin) were significantly more potent inhibitors of (α4)2(ß2)3 nAChRs than of (α4)3(ß2)2 nAChRs. This stoichiometry-selective effect was most pronounced with pefloxacin, which inhibited (α4)2(ß2)3 nAChRs with an IC50 of 26.4 ± 3.4 µM but displayed no significant inhibition of (α4)3(ß2)2 nAChRs. In contrast, two nonfluorinated quinolone antibiotics (cinoxacin and oxolinic acid) exhibited no selectivity in their inhibition of the two stoichiometries of α4ß2. Computational docking studies suggest that pefloxacin interacts selectively with an allosteric transmembrane site at the ß2(+)/ß2(-) subunit interface, which is consistent with its selective inhibition of (α4)2(ß2)3. These findings concerning the antagonist effects of fluoroquinolones provide further evidence that differences in the subunit stoichiometry of heteromeric nAChRs can result in substantial differences in pharmacological properties.

TEMPy2: a Python library with improved 3D electron microscopy density-fitting and validation workflows.

Structural determination of molecular complexes by cryo-EM requires large, often complex processing of the image data that are initially obtained. Here, TEMPy2, an update of the TEMPy package to process, optimize and assess cryo-EM maps and the structures fitted to them, is described. New optimization routines, comprehensive automated checks and workflows to perform these tasks are described.

Automated Modeling and Validation of Protein Complexes in Cryo-EM Maps.

The resolving power of cryo-EM experiments has dramatically improved in recent years. However, many cryo-EM maps may still not achieve a resolution that is sufficiently high to allow model building directly from the map. Instead, it is common practice to fit an initial atomic model to the map and refine this model. Depending on the resolution and whether the structure suffers from inherent flexibility or experimental limitations, different methods can be applied, to obtain high-quality, well-fitted atomic model of the macromolecular assembly represented by the map, and to assess its properties. In this review, we describe some of these methods, with the main focus on those that have been developed in our group over the last decade.

Structure of Microtubule-Trapped Human Kinesin-5 and Its Mechanism of Inhibition Revealed Using Cryoelectron Microscopy.

Kinesin-5 motors are vital mitotic spindle components, and disruption of their function perturbs cell division. We investigated the molecular mechanism of the human kinesin-5 inhibitor GSK-1, which allosterically promotes tight microtubule binding. GSK-1 inhibits monomeric human kinesin-5 ATPase and microtubule gliding activities, and promotes the motor's microtubule stabilization activity. Using cryoelectron microscopy, we determined the 3D structure of the microtubule-bound motor-GSK-1 at 3.8 Å overall resolution. The structure reveals that GSK-1 stabilizes the microtubule binding surface of the motor in an ATP-like conformation, while destabilizing regions of the motor around the empty nucleotide binding pocket. Density corresponding to GSK-1 is located between helix-α4 and helix-α6 in the motor domain at its interface with the microtubule. Using a combination of difference mapping and protein-ligand docking, we characterized the kinesin-5-GSK-1 interaction and further validated this binding site using mutagenesis. This work opens up new avenues of investigation of kinesin inhibition and spindle perturbation.

The initial stage of structural transformation of Aß42 peptides from the human and mole rat in the presence of Fe2+ and Fe3+: Related to Alzheimer's disease.

The early stage of secondary structural conversion of amyloid beta (Aß) to misfolded aggregations is a key feature of Alzheimer's disease (AD). Under normal physiological conditions, Aß peptides can protect neurons from the toxicity of highly concentrated metals. However, they become toxic under certain conditions. Under conditions of excess iron, amyloid precursor proteins (APP) become overexpressed. This subsequently increases Aß production. Experimental studies suggest that Aß fibrillation (main-pathway) and amorphous (off-pathway) aggregate formations are two competitive pathways driven by factors such as metal binding, pH and temperature. In this study, we performed molecular dynamic (MD) simulations to examine the initial stage of conformational transformations of human Aß (hAß) and rat Aß (rAß) peptides in the presence of Fe2+ and Fe3+ ions. Our results demonstrated that Fe2+ and Fe3+ play key roles in Aßs folding and aggregation. Fe3+ had a greater effect than Fe2+on Aßs' folding during intermolecular interactions and subsequently, had a greater effect in decreasing structural diversity. Fe2+ was observed to be more likely than Fe3+ to interact with nitrogen atoms from the residues of imidazole rings of His. rAß peptides are more energetically favorable than hAß for intermolecular interactions and amorphous aggregations. We concluded that most hAß structures were energetically unfavorable. However, hAßs with intermolecular ß-sheet formations in the C-terminal were energetically favorable. It is notable that Fe2+ can change the surface charge of hAß. Furthermore, Fe3+ can promote C-terminal folding by binding to Glu22 and Ala42, and by forming stable ß-sheet formations on the C-terminal. Fe3+ can also pause the main-pathway by inducing random aggregations.

Diazepam-induced loss of inhibitory synapses mediated by PLCδ/ Ca2+/calcineurin signalling downstream of GABAA receptors.

Benzodiazepines facilitate the inhibitory actions of GABA by binding to γ-aminobutyric acid type A receptors (GABAARs), GABA-gated chloride/bicarbonate channels, which are the key mediators of transmission at inhibitory synapses in the brain. This activity underpins potent anxiolytic, anticonvulsant and hypnotic effects of benzodiazepines in patients. However, extended benzodiazepine treatments lead to development of tolerance, a process which, despite its important therapeutic implications, remains poorly characterised. Here we report that prolonged exposure to diazepam, the most widely used benzodiazepine in clinic, leads to a gradual disruption of neuronal inhibitory GABAergic synapses. The loss of synapses and the preceding, time- and dose-dependent decrease in surface levels of GABAARs, mediated by dynamin-dependent internalisation, were blocked by Ro 15-1788, a competitive benzodiazepine antagonist, and bicuculline, a competitive GABA antagonist, indicating that prolonged enhancement of GABAAR activity by diazepam is integral to the underlying molecular mechanism. Characterisation of this mechanism has revealed a metabotropic-type signalling downstream of GABAARs, involving mobilisation of Ca2+ from the intracellular stores and activation of the Ca2+/calmodulin-dependent phosphatase calcineurin, which, in turn, dephosphorylates GABAARs and promotes their endocytosis, leading to disassembly of inhibitory synapses. Furthermore, functional coupling between GABAARs and Ca2+ stores was sensitive to phospholipase C (PLC) inhibition by U73122, and regulated by PLCδ, a PLC isoform found in direct association with GABAARs. Thus, a PLCδ/Ca2+/calcineurin signalling cascade converts the initial enhancement of GABAARs by benzodiazepines to a long-term downregulation of GABAergic synapses, this potentially underpinning the development of pharmacological and behavioural tolerance to these widely prescribed drugs.

Influence of Exercise on Patients with Guillain-Barré Syndrome: A Systematic Review.

Purpose: To evaluate the effects of exercise interventions on improving physical outcomes in patients with Guillain-Barré syndrome (GBS). Methods: The PubMed database was searched for articles published up to and including February 2015. Randomized controlled trials (RCTs), case reports, and quasi-experimental and single-subject designs published in English-language, peer-reviewed journals that assessed the impact of physical exercise on patients with GBS were included; study quality was assessed using Sackett's rules of evidence. Data are presented qualitatively and quantitatively using numerical values and percentages. Results: Seven articles were included in the systematic review. One RCT showed that high-intensity relative to lower intensity exercise significantly reduced disability in patients with GBS, as measured with the FIM (p<0.005, r=0.71). Overall, various types of exercise programmes improve physical outcomes such as functional mobility, cardiopulmonary function, isokinetic muscle strength, and work rate and reduce fatigue in patients with GBS. Conclusion: Because of insufficient high-quality literature, making confident conclusions about the effects of exercise interventions on physical outcomes in patients with GBS is not possible. Future research should consider using higher quality study designs to confirm the results outlined in this article.

Objectif : évaluer les effets des programmes d'exercice sur l'amélioration des capacités physiques des patients atteints du syndrome de Guillain-Barré (SGB). Méthode : une recherche a été effectuée dans la base de données PubMed sur les articles publiés jusqu'en février 2015. Des essais contrôlés randomisés (ECR), des études de cas, des modèles quasi expérimentaux et des études individuelles publiés dans des revues anglophones révisées par les pairs qui évaluaient l'effet de l'exercice physique sur les patients atteints du SGB ont été inclus; la qualité des études a été évaluée à l'aide de la règle des preuves de Sackett. Les données ont été présentées de manière qualitative et quantitative à l'aide de valeurs numériques et de pourcentages. Résultats : sept articles ont été inclus dans la revue systématique. Un ECR a montré que l'exercice à haute intensité par rapport à l'exercice à faible intensité réduisait de manière significative les incapacités des patients atteints du SGB, mesurées par la mesure d'indépendance fonctionnelle (p<0,005, r=0,71). Globalement, divers types de programmes d'exercice améliorent les capacités physiques, dont la mobilité fonctionnelle, la fonction cardiopulmonaire, la force musculaire isocinétique et le rythme de travail, et réduisent la fatigue chez les patients atteints du SGB. Conclusion : en raison du manque d'études de qualité, il n'est pas possible de tirer de conclusions définitives sur les effets des programmes d'exercice sur les capacités physiques des patients atteints du SGB. Des recherches futures se fondant sur des modèles d'études de qualité supérieure devraient être envisagées pour confirmer les résultats présentés dans cette revue.

Reclassification of cardiovascular risk in patients with normal myocardial perfusion imaging using heart rate response to vasodilator stress.

Previous studies have shown that patients with normal vasodilator myocardial perfusion imaging (MPI) findings remain at a greater risk of future cardiac events than patients with normal exercise MPI findings. The aim was to assess improvement in risk classification provided by the heart rate response (HRR) in patients with normal vasodilator MPI findings when added to traditional risk stratification. We retrospectively studied 2,000 patients with normal regadenoson or adenosine MPI findings. Risk stratification was performed using Adult Treatment Panel III framework. Patients were stratified by HRR (percentage of increase from baseline) into tertiles specific to each vasodilator. All-cause mortality and cardiac death/nonfatal myocardial infarction (MI) ≤2 years from the index MPI were recorded. During follow-up, 11.8% patients died and 2.7% patients experienced cardiac death/nonfatal MI in the adenosine and regadenoson groups, respectively. The patients who died had a greater Framingham risk score (12 ± 4 vs 11 ± 4, p = 0.009) and lower HRR (22 ± 16 vs 32 ± 21, p <0.0001). In an adjusted Cox model, the lowest tertile HRR was associated with an increased risk of mortality (hazard ratio 2.1) and cardiac death/nonfatal MI (hazard ratio 2.9; p <0.01). Patients in the highest HRR tertile, irrespective of the Adult Treatment Panel III category, were at low risk. When added to the Adult Treatment Panel III categories, the HRR resulted in net reclassification improvement in mortality of 18% and cardiac death/nonfatal MI of 22%. In conclusion, a blunted HRR to vasodilator stress was independently associated with an increased risk of cardiac events and overall mortality in patients with normal vasodilator MPI findings. The HRR correctly reclassified a substantial proportion of these patients in addition to the traditional risk classification models and identified patients with normal vasodilator MPI findings, who had a truly low risk of events.

How much alcohol should we infuse in the coronary artery of hypertrophic cardiomyopathy patients?

BACKGROUND: Although alcohol septal ablation (ASA) is increasingly used in hypertrophic cardiomyopathy (HC) patients who are refractory to medical therapy, the amount of alcohol that is required has not been well studied. This study sought to determine the amount of alcohol that is necessary to achieve clinical benefits of ASA. METHODS: Myocardial perfusion imaging was used to determine the size of the myocardial infarction produced by ASA in 54 HC patients. Left ventricular outflow gradients (LVOTg) were determined invasively before and after ASA and by Doppler echocardiography before and at a median of 3 months after ASA. RESULTS: LVOTg decreased at rest and after provocation in response to ASA and this was maintained on follow-up at 3 months. There was no relationship between the amount of alcohol infused and the infarct mass as determined by myocardial perfusion imaging. While the infarct mass was not correlated with the drop in the LVOTg at rest or with provocation, the quantity of alcohol infused was correlated with the drop in LVOTg at rest (r = 0.27, p = 0.05) and with provocation (r = 0.34, p = 0.02). Furthermore, infusing more than 2ml of absolute alcohol was associated with a drop in the LVOTg by more than 60 mmHg at rest (p = 0.02) and by more than 130 mmHg with provocation (p = 0.05). CONCLUSIONS: Although lower amounts of alcohol infusion are desirable to avoid side-effects, it might be prudent to infuse around 2 ml of absolute alcohol in order to achieve the desirable degree of LVOTg reduction in ASA.